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History and Development of the Polymerase Chain Reaction (PCR)

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Polymerase Chain Reaction - PCR

Table of Contents:

Introduction to PCR - Polymerase Chain Reaction 

Polymerase Chain Reaction (PCR)

PCR, a Concept to be Discovered

General Principles of the PCR

Polymerases/Reaction Specificity and Efficiency

Utility of PCR

PCR and Molecular Cloning

Misincorporation: Errors of In Vitro Systems

Reaction Specificity

Major Advantages of PCR as a Cloning Method include its Rapidity, Sensitivity, and Robustness

Limitations of PCR

Instruments for PCR

Oligonucleotide Synthesis

Primer Design

PCR Today

Will PCR ever be replaced? – Helicase-dependent Amplification (HDA)

Introduction to PCR - Polymerase Chain Reaction 

      More than 30 years ago, the introduction of recombinant DNA technology as a tool for the biological sciences revolutionized the study of life.  Molecular cloning allowed the study of individual genes of living organisms; however this technique was dependent on obtaining a relatively large quantity of pure DNA.  This depended on the replication of the DNA of plasmids or other vectors during cell division of microorganisms (1).  Researchers found it extremely laborious and difficult to obtain a specific DNA in quantity from the mass of genes present in a biological sample (2).   Recombinant DNA technology made possible the first molecular analysis and prenatal diagnosis of several human diseases.  Fetal DNA obtained by amniocentesis sampling could be analyzed by restriction enzyme digestion, electrophoresis, southern transfer and hybridization to a cloned gene or oligonucleotide probes (3).  However, southern blotting permitted only rudimentary mapping of genes in unrelated individuals (4). 

Polymerase Chain Reaction (PCR)


PCR, an acronym for Polymerase Chain Reaction (5,6), allowed the production of large quantities of a specific DNA from a complex DNA template in a simple enzymatic reaction.  PCR is a recently developed procedure for the in vitro amplification of DNA.  PCR has transformed the way that almost all studies requiring the manipulation of DNA fragments may be performed as a results of its simplicity and usefulness (7). 
In the 1980s, Kary Mullis (Figure 1) and a team of researchers at Cetus Corporation at Cetus Corporation conceived of a way to start and stop a polymerase's action at specific points along a single strand of DNA.  Mullis also realized that by harnessing this component of molecular reproduction technology, the target DNA could be exponentially amplified.  This DNA amplification procedure was based on an in vitro rather than an in vivo process (5,6,8).  Cell-free DNA amplification by PCR was able to simplify many of the standard procedures for cloning, analyzing, and modifying nucleic acids (1).  Previous techniques for isolating a specific piece of DNA relied on gene cloning – a tedious and slow procedure.  PCR, on the other hand Kerry Mullis stated “lets you pick the piece of DNA you’re interested in and have as much of it as you want” (2,8).   When other Cetus scientists eventually succeeded in making the polymerase chain reaction perform as desired in a reliable fashion, they had an immensely powerful technique for providing essentially unlimited quantities of the precise genetic material molecular biologists and others required for their work (8).  Since the first report in1985, more than 5000 scientific papers were published by 1992 (1).  Furthermore, the large number of publications of course makes it impossible to review all the important contributions to the development and application of PCR technology; however we will attempt to review here the most important developments in the practice of basic PCR. 

PCR, a Concept to be Discovered


PCR was thought to be conceived by Dr. Kerry Mullis in 1983 while working at the Cetus Corporation in Emeryville, CA.  However, some pioneering work was also done by Gobind Khorana in 1971 who described a basic principle of replicating a piece of DNA using two primers.  Progress then was limited by primer synthesis and polymerase purification issues (9).   In Mullis’s head, the invention grew from a theoretical scheme to perform limited dideoxynucleotide sequencing of unique human genes using synthetic oligonucleotides for the purpose of diagnosing common human disease mutations.  An obvious obstacle to such a direct sequencing strategy was the high complexity of the human genome (3.3 X 109 base pairs).  Thus, a second oligonucleotide or primer was added to block the progression of the synthesis of the first primer.   Later however, this second primer was included to bind to the other DNA strand, so that each strand of the mutant allele would contribute to the eventual signal.  If the scheme involving simultaneous hybridization of primers to each strand was modified by heating the mixture and then repeating the annealing and extension steps, then the primary signal would be increased even further.  Repeating the steps would enable the products of the first round to be duplicated in the second cycle, to yield two copies.  Repeating the cycle again would result in four copies, et cetera.  Several weeks passed before this great idea was attempted (8).  Two primers were synthesized to be perfectly complementary to each end of the 110 base pair region of a cloned segment of the human b-globin gene, the amplification was performed, and the products were identified by acrylamide gel electrophoresis.  The end result was the anticipated 110 base pair DNA fragment and the beginning of PCR as a basic technique in molecular biology (5,6).
In Mullis's original PCR process(5,6,8), the enzyme was used in vitro (in a controlled environment outside an organism).  The double-stranded DNA was separated into two single strands by heating it to 96°C.  At this temperature, however, the E.Coli DNA polymerase was destroyed so that the enzyme had to be replenished after the heating stage of each cycle.  Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process.

General Principles of the PCR


      Examination of the PCR amplification mechanism reveal its simplicity but also its elegance (Figure 2).  Oligonucleotide primers are first designed to be complementary to the ends of the sequence to be amplified, and then mixed in molar excess with the DNA template and deoxyribonucleotides in an appropriate buffer.  Following heating to denature the original strands and cooling to promote primer annealing, the oligonucleotides each bind to a different strand of the target fragment.  The primers are positioned so that when each is extended by the action of a DNA polymerase, the newly synthesized strands will overlap the binding site of the opposite oligonucleotide.  As the process of denaturation, annealing, and polymerase extension is continued the primers repeatedly bind to both the original DNA template and complementary sites in the newly synthesized strands and are extended to produce new copies of DNA (Figure 3).  The end result is an exponential increase in the total number of DNA fragments that include the sequences between the PCR primers, which are finally represented at a theoretical abundance of 2n, where n is the number of cycles (1,7,13).

Polymerases/Reaction Specificity and Efficiency


A DNA polymerase is a naturally occurring enzyme, a biological macromolecule that catalyzes the formation and repair of DNA. It works by binding to a single DNA strand and creating a complementary strand.  The accurate replication of all living matter depends on this activity, where it functions to duplicate DNA when cells divide (10,11).  Only recently have scientists learned to manipulate this activity and apply it to scientific research.  The earliest PCR experiments utlilized the Klenow fragment of Escherichia coli DNA polymerase I at a temperature of 37C to amplify specific targets from human genomic DNA (5,6).  Often these PCR reactions produced incompletely pure target product as judged by gel electrophoresis (1).  These initial PCR amplifications with the Klenow fragment were not highly specific (5,6).  Although a unique DNA fragment could be amplified ~200,000 fold from genomic DNA, only about 1% of the PCR product was the targeted sequence (13).  A specific hybridization probe was required to analyse the amplified DNA (5,6).  
Some PCR conditions were determined to increase the stringency of primer hybridization such as lower MgCl2 concentrations and higher annealing temperatures.
Furthermore, the concentration of enzyme and primers, the annealing time, extension time, and number of PCR cycles all were found to effect the specificity of the PCR.
Also, the concentration of a specific sequence in a sample can also influence the relative homogeneity of the PCR products (1,7,13,14,15).   Deoxyribonucleotide triphosphates and magnesium in an appropriate buffer are also important ingredients for PCR.  The efficiency and specificity of PCRs can be affected by variations in the concentration and ratio of free magnesium, deoxyribonucleotide triphosphates, and primers.  These reagents must be optimized in order to achieve high specificity and yield (14).  It was also discovered that the effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction (15).
The inactivation of the Klenow fragment of Escherichia coli DNA polymerase I at the high temperature required for strand separation required the addition of enzyme after the denaturation step of each cycle (5,6).  Prior to 1988, anyone conducting a PCR reaction procedure was obliged to sit patiently by a series of water baths or heating blocks and add a fresh aliquot of E.Coli DNA polymerase after each denaturation step, which was typically carried out by immersing the reaction vessel in boiling water for ½ a minute to 3 minutes (7).   This rather tedious step was eliminated by the introduction of a thermostable DNA polymerase, the Taq DNA polymerase (12) once, at the beginning of the PCR reaction.  The thermostable properties of the DNA polymerase activity were isolated from Thermus aquaticus (Taq) (Figure 4) that grow in geysers of over 110C, and have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction (1,7,12).   The Taq enzyme can withstand repeated heating to 94C and so each time the mixture is cooled to allow the oligonucleotide primers to bind the catalyst for the extension is already present (1,7).  However, higher annealing temperatures were not established until the single “most important development of PCR development” (8), the purification and commercial distribution of a heat-resistant DNA polymerase from the thermophilic bacterium Thermus aquaticus (Taq) (12). 
The isolation of a heat-resistant DNA polymerase also allowed primer annealing and extension to be carried out at elevated temperatures (1,7,12,13), thereby reducing mismatched annealing to nontarget sequences (non-specific amplification) or increasing specificity.   In this way, for many amplifications the PCR product could be detected as a single ethidium bromide-stained band on an electrophoretic gel (12).  This increased specificity also increased DNA yield of the target sequence.  Moreover, longer PCR products could be amplified from genomic DNA, probably due to a reduction in the secondary structure of the template strands at the elevated temperature used for primer extension.  The upper size limit for Klenow fragment polymerase amplification was only about 400bp.  Taq polymerase and other thermostable polymerases have synthesized fragments up to 10 kb (1,7,12,13).  The availability of Taq polymerase has also greatly simplified the automation of the reaction as it is a much easier task to construct an apparatus that will cycle a reaction tube through different temperatures than to manufacture a device that would perform both the thermocycling and the addition of enzyme aliquots.  Currently there is a great variety of thermocyclers available commercially.  This development has been a significant factor in the rapid application of this technology by the scientific community (7). 

Utility of PCR


      In addition to the production of double-stranded, blunt-ended DNA fragments which may be formed by PCR, two other features of the PCR scheme contribute greatly to the utility of PCR.  First, the position of binding of the primers defines the boundaries of the amplified fragment and therefore the prior molecular cloning requirement of restriction endonuclease recognition sites is not required for PCR.  As only a limited number of DNA sequences are restriction sites, PCR greatly increases the flexibility of choice of fragment size and composition.  Secondly, it is not necessary for PCR oligonucleotides to be exactly complementary to the template DNA.  “Tails” may be added to the 5’ end of the primer to introduce sequences within the priming sites which thus may be exploited to introduce restriction endonuclease recognition sites or other useful sequences such as mutations into the amplified DNA.  This phenomena allowed the emergence of PCR as a method for rapid DNA cloning (1,7,13).

PCR and Molecular Cloning


Molecular cloning has benefited from the emergence of PCR as a technique.  Direct cloning was first conducted using a 110 bp DNA fragment amplified by PCR and oligonucleotide primers which contained restriction endonuclease recognition sites added to their 5’ ends.  These sites were used to facilitate cloning of the amplified DNA into an M13 plasmid (17).  The 110 bp fragment was also sequenced to confirm that this approach was a rapid yet reliable approach to cloning.  (Figure 5)

Misincorporation: Errors of In Vitro Systems


Cell-based DNA cloning involves DNA replication in vivo, which is associated with a very high fidelity of copying because of proofreading mechanisms.  However, when DNA is replicated in vitro as with PCR, the copying error rate is considerably greater.  The most widely used polymerase, Taq DNA polymerase however, has no associated 3’to 5’ exonuclease to confer a proofreading function.  Thus the error rate due to base misincorporation during DNA replication is rather high for Taq: for a 1 kb sequence that has undergone 20 effective cycles of duplication, approximately 40% of the new DNA strands synthesized by PCR using this enzyme will contain an incorrect nucleotide resulting from a copying error (16).  Therefore, even if the PCR reaction involves amplification of a single DNA sequence, the final product will be a mixture of almost matching, but not identical DNA sequences.  Despite the errors due to replication in vitro, DNA sequencing of the total PCR product may give the correct sequence due to the fact that the incorporation of incorrect bases is essentially random and the contribution of one incorrect base on one or more strands is overwhelmed by the contributions from the huge majority of strands which will have the correct sequence.  However, if the PCR product is to be cloned in cells, several individual clones may need to be sequenced in order to determine the correct (consensus) sequence, prior to conducting further experiments.
More recently, the problem of infidelity of DNA replication during the PCR reaction has been considerably reduced by using alternative heat-stable DNA polymerases which have associated 3’ to 5’ exonuclease activity.  Pyrococcus furiosus (Pfu) DNA polymerases and Thermococcus Litoralis (VENT) are becoming more widely used because of the proofreading conferred by their associated 3’ to 5’ exonuclease activity (18).  The resulting PCR product of Pfu for example, has a much lower level of mutations introduced by copying errors: for a 1 kb segment of DNA that has undergone 20 effective cycles of duplication, about 3.5% of the DNA strands in the product carry an altered base (16).

Reaction Specificity


New approaches to improve specificity have been developed based on the recognition that the Taq DNA polymerase retains considerable enzymatic activity at temperatures well below the optimum for DNA synthesis.  Thus, primers annealing non-specifically to a partially single stranded template region can be extended before the reaction reaches 72°C for extension of specifically annealed primers.  If the DNA polymerase is activated only after the reaction has reached high (>70°C) temperatures, non-target amplification can be minimized (19,20).  This “Hot start” approach can be accomplished by manual addition of an essential reagent to the selection tube at elevated temperatures.  The addition of ssDNA binding protein has also been reported to increase specific amplification.  A more user friendly approach is to use either inhibition or inactivation of the DNA polymerase itself.  Two types of inhibition of Taq DNA polymerase have been tried including oligonucleotide inhibition (21) and antibody (22) inhibition. Highly specific oligonucleotide inhibitors of both Taq DNA polymerases have been produced.  These selectively inhibit DNA polymerase activity at temperatures below 40°C and have been shown to function in Hot Start applications.  Alternatively, one can use an antibody against Taq DNA polymerase. The antibody inhibits the DNA polymerase until the temperature of the PCR is such that the antibody is denatured at a temperature greater than 55°C, thereby releasing the enzyme.  However there are disadvantages to this type of Hot Start conditions.  In this case, one needs an antibody for each different enzyme used in a PCR and for a large number of PCRs this can rise costs significantly.  The most convenient form of Hot Start is to modify the DNA polymerase in such a way that it is inactive at room temperature (temperature-sensitive mutant), and is only re-activated following incubation at 95°C for 6-15 minutes (23).  

Major Advantages of PCR as a Cloning Method include its Rapidity, Sensitivity, and Robustness



Because of its simplicity, PCR is a popular technique with a wide range of applications
including direct sequencing, genomic cloning, DNA typing, detection of infectious microorganisms, site-directed mutagenesis, prenatal genetic disease research, and analysis of allelic sequence variations (1,7,13,16) which depend on essentially three major advantages of the method:


Speed and ease of use:   DNA cloning by PCR can be performed in a relatively short amount of time, within a few hours.  Usually, a PCR reaction consists of around 30 cycles each cycle containing a denaturation, synthesis and reannealing step, with an individual cycle typically taking 35 min in an automated thermal cycler.  This is clearly quicker than the time required for cell-based DNA cloning, which could take weeks of time.  Furthermore, it is quite easy to setup a PCR reaction and the use of a thermocycler machine is also easy.  Some time is required for the design and synthesis of oligonucleotide primers, but this has been simplified by the availability of computer software for primer design and rapid commercial or academic synthesis of custom oligonucleotides.   Optimization of PCR conditions may be required such as primer annealing temperature, magnesium concentration, and primer concentration.  However, the creation of gradient PCR machines which allow a variety of primer annealing temperatures to be tested at the same time has greatly decreased the time required for this step.  Once the optimal conditions for a reaction have been obtained, the reaction can then be simply repeated (1,7,13,16). 


Sensitivity:  PCR is capable of amplifying sequences from minute amounts of target DNA, even the DNA from a single cell (24).  Such exquisite sensitivity has afforded new methods of studying molecular pathogenesis and has found numerous applications in forensic science, in diagnosis, in genetic linkage analysis using single-sperm typing and in molecular paleontology studies, where samples may contain minute numbers of cells. However, the extreme sensitivity of the method means that great care has to be taken to avoid contamination of the sample under investigation by external DNA, such as from minute amounts of cells from the operator (1,7,13,16).


Robustness:  A broad range of nucleic acid sources are suitable templates for PCR amplification.  Purified DNAs from various species and sources have been amplified.  PCR can permit amplification of specific sequences from material in which the DNA is badly degraded or embedded in a medium from which conventional DNA isolation is problematic. As a result, it is again very suitable for molecular anthropology and paleontology studies, for example the analysis of DNA recovered from archaeological remains. It has also been used successfully to amplify DNA from formalin-fixed or paraffin-embedded tissue samples, which has important applications in molecular pathology and, in some cases, genetic linkage studies.  Generally, the success of PCR amplification is greatest when target fragments are relatively abundant (1,7,13,16). 

Limitations of PCR


Despite its huge popularity, PCR has certain limitations as a method for selectively cloning specific DNA sequences. 
In order to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence, some prior sequence information is usually necessary.  This normally means that the DNA region of interest has been partly characterized previously, often following prior cell-based DNA cloning.  However, a variety of approaches have been developed that reduce or even exclude the need for prior DNA sequence information concerning the target DNA.  Previously uncharacterized DNA sequences can sometimes be cloned using PCR with degenerate oligonucleotides if they are members of a gene or repetitive DNA family at least one of whose members has previously been characterized.   In some cases, PCR can be used effectively without any prior sequence information concerning the target DNA to permit indiscriminateamplification of DNA sequences from a source of DNA that is present in extemely limited quantities.  Therefore, although PCR can be applied to ensure whole genome amplification, it does not have the advantage of cell-based DNA cloning in offering a way of separating the individual DNA clones comprising a genomic DNA library.
The amount of PCR product obtained in a single reaction is also much more limited than the amount that can be obtained using cell-based cloning where scale-up of the volumes of cell cultures is possible. The efficiency of a PCR reaction will vary from template to template and according to various factors that are required to optimize the reaction but typically only comparatively small amounts of product are achieved.
Although the theoretical yield of PCR is exponential, the actual yield of a PCR is much less indicating that the scheme is operating with less than its maximum potential.  For example, the amount of product at each cycle eventually levels off.  This plateau may be explained by the following phenomena.  First, some of the template may never be available due to strand breaks or failure of the DNA to dissociated from other macromolecules during purification and the initial thermocycles.  Secondly, the amount of enzyme is finite and eventually activity may decrease.  Thirdly, as the concentration of the double-stranded product reaches high levels, competition increases between annealing of template (PCR product) to primer and reannealing of the complementary template strands (1,7,13).
An obvious and many times great disadvantage of PCR as a DNA cloning method has been the size range of the DNA sequences that can be cloned.  Unlike cell-based DNA cloning where the size of cloned DNA sequences can approach 2 Mb, reported DNA sequences cloned by PCR have typically been in the 0.15 kb size range, often at the lower end of this scale.  Small fragments of DNA can usually be amplified easily by PCR, however it becomes increasingly more difficult to obtain efficient amplification as the desired product length increases.  Barnes (25) recognized a target length limitation to PCR amplification of DNA.  He used a combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent) to conduct high fidelity long PCR. At least 35 kb of bacteriophage lambda can be amplified to high yields from 1 ng of lambda DNA template.  Use of this method yielded increased base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment.  Other conditions have been identified for effective amplification of longer targets, including amplification of up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA (26).  The conditions for these long PCRs included increased pH, addition of glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and the use of a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity.  The "long PCR" protocol maintained the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing.  The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics (26).  Generally, the conditions for long range PCR involve a combination of modifications to standard conditions with a two-polymerase system.  This provides optimal levels of DNA polymerase and 3’to 5’ exonuclease activity which serves as a proofreading mechanism (16).

Instruments for PCR


Thermocyclers which automatically regulate temperatures for PCR cycling were introduced in 1986 (Figure 6).  In addition to the advances in PCR reagents, new instruments for automated thermal cycling and for analyzing PCR products have been developed.  New thermal cyclers have increased rates of heating, cooling, and heat transfer to modified reaction vessels.  The reaction vessels accommodated by the first generation thermal cyclers (or even water baths and heating blocks) were standard plastic microfuge tubes.  PCR amplification in thin capillary tubes allowed rapid thermal cycling, and DNA synthesis to 20s.  The speed of the temperature changes achieved in these systems has allowed the precise definition of temperature optima for each individual step in the PCR cycle.   The new generation thermal cyclers also accommodate more samples, have more precise thermal profiles, and are programmable (13). 

Oligonucleotide Synthesis


One of the least appreciated contributions to the widespread application of PCR has been the development of reliable automated chemistry for oligonucleotide synthesis.  Until recently, the construction of a single oligonucleotide was a substantial task that could only be performed by a skilled organic chemist.  Now it is possible to purchase either an oligonucleotide synthesizer that can be operated by a technician or the oligonucleotides themselves from a commercial or academic source.  Multiplex oligonucleotide synthesis machines have been constructed with the aim of reducing the overall cost of synthesis (27,28).  As the oligonucleotides define the eventual PCR products, there is little doubt that in the absence of their ready supply, PCR would not have enjoyed the wide acceptance that it has gained today (13). 

Primer Design


Researchers agreed early on that the design of PCR primers was difficult and unreliable.  Computer programs were devised to take all of the design criteria into account.   One of the first programs written for primer design was Olga which made use of the implementation of Digital Research GEM (Graphics Environment Manager) on the Atari ST (29).  Olga was specifically suited to the polymerase chain reaction (PCR) allowing simultaneous analysis of two primer sequences.  The advantage of Olga was that it provided in one program analyses for direct repeats, secondary structures and primer dimerization as well as several useful 'finishing' tools for workers engaged in PCR optimization and oligonucleotide syntheses.   The Primer3 program at the Whitehead Institute is now thought to be the most reliable and versatile tool currently available (30).

PCR Today


PCRs can now be performed enabling the amplification of DNA fragments up to several kilobases in length by more than one million times their initial abundance.  The procedure is highly automatable and requires just a few hours from beginning the thermocyling to product analysis.  This was not the case previously, and the practical requirements for performing a PCR have been greatly simplified since the first manuscripts of the method (13).  Today, most of the initial hitches or inefficiencies of the PCR have been worked out (8).   Furthermore, PCR has expanded to include more than 270,000 articles (31).

Will PCR ever be replaced? – Helicase-dependent Amplification (HDA)


  Polymerase chain reaction is the most widely used method for in vitro DNA amplification however it requires thermal denaturation or thermocycling to separate the two DNA strands.  In vivo, DNA is replicated by DNA polymerases with various accessory proteins.   DNA helicase, a DNA polymerase accessory proteins acts to separate duplex DNA inside cells.  Vincent et al. (32) have devised a new in vitro isothermal DNA amplification method by mimicking the in vivo replication mechanism.  Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization.  Subsequent primer extension is then catalyzed by a DNA polymerase.  HDA does not require an expensive thermocycler and thus PCR may be performed practically anywhere.  In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. HDA offers great promise in the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care (32).

Conclusions


It is said the simplest and most convenient way to define PCR is as a technique.  However, such a categorization eliminates the history of PCR's development as many individuals over the years contributed to the ideas behind the theory of PCR and the fine-tuning of the technique.  The next simplest answer is to name an individual as the inventor of the polymerase chain reaction.  Karry Mullis was awarded the Nobel Prize for Chemistry in 1993 for his discovery of PCR.  However, this discovery is contested amongst many scientists, all of which may have contributed to unlocking this puzzle.
It has also been said that PCR did not exist until it was made to work in an experimental system.  With this in mind, merely the thought of a concept is not sufficient; a concept must have been successfully been put into practice (33).
Although there is doubt as to the ultimate creator of PCR, and doubt as to the possibility that PCR may somehow or sometime be replaced, there is little doubt the impact that PCR has created over a short time span on the study of molecular biology and life.

References

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27.       Caruthers MH, Barone AD, Beaucage SL, Dodds DR, Fisher EF, McBride LJ, Matteucci M, Stabinsky Z, Tang JY.  Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method.  Methods Enzymol. 1987;154:287-313.
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31. Location world wide web: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed Search: “PCR”
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33. Paul Rabinow. Making PCR, A Story of Biotechnology, University of Chicago Press, 1996

 

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